The present invention relates to methods and compositions for detecting splicing defects in the dihydropyrimidine dehydrogenase gene. The methods and compositions are useful for identifying persons who are at risk of a toxic reaction to the commonly employed cancer chemotherapy agent, 5-fluorouracil.
5-Fluorouracil (5-FU) is commonly used in the treatment of cancers, including cancers of the breast, head, neck, and digestive system. The efficacy of 5-FU as a cancer treatment varies significantly among patients. Clinically significant differences in systemic clearance and systemic exposure of 5-FU are often observed. See, Grem, J. L. In Chabner, B. A. and J. M. Collins (eds.), Cancer Chemotherapy: Principles and Practice, pp. 180-224, Philadelphia, Pa., Lippincott, 1990). Furthermore, 5-FU treatment is severely toxic to some patients, and has even caused death. See, Fleming et al. (1993) Ear. J. Cancer 29A: 740-744; Thyss et al. (1986) Cancer Chemother. Pharmacol. 16: 64-66; Santini et al. (1989) Br. J. Cancer 59: 287-290; Goldberg et al. (1988) Br. J. Cancer 57: 186-189; Trump et al. (1991) J. Clin. Oncol. 9: 2027-2035; and Au et al. (1982) Cancer Res. 42: 2930-2937.
Patients in whom 5-FU is severely toxic typically have low levels of dihydropyrimidine dehydrogenase (DPD) activity. See, Tuchman et al. (1985) N. Engl. J. Med. 313: 245-249; Diasio et al. (1988) J. Clin. Invest. 81: 47-51; Fleming et al. (1991) Proc. Am. SAc. Cancer Res. 32: 179; Harris et al. (1991) Cancer (Phila.) 68: 499-501; Houyau et al. (1993) J. Nat""l. Cancer Inst. 85: 1602-1603; Lyss et al. (1993) Cancer Invest. 11: 239-240. Dihydropyrimidine dehydrogenase. (DPD, EC 1.3.1.2) is the principal enzyme involved in the degradation of 5-FU, which acts by inhibiting thymidylate synthase. See, Heggie et al (1987) Cancer Res. 47: 2203-2206; Chabner et al. (1989) In DeVita et al. (ads.), Cancerxe2x80x94Principles and Practice of Oncology, pp. 349-395, Philadelphia, Pa., Lippincott; Diasio et al. (1989) Clin. Pharmacokinet. 16: 215-27; Grem et. al., supra. The level of DPD activity also affects the efficacy of 5-FU treatments, as 5-FU plasma levels are inversely correlated with the level of DPD activity. See, Iigo et al. (1988) Biochem, Pharm. 37: 1609-1613; Goldberg et al., supra.; Harris et al., supra.; Fleming et al., supra. In turn, the efficacy of 5-FU treatment of cancer is correlated with plasma levels of 5-FU.
In addition to its 5-FU degrading activity, DPD is also the initial and rate limiting enzyme in the three-step pathway of uracil and thymine catabolism, leading to the formation of xcex2-alanine and xcex2-aminobutyric acid, respectively. See, Wasternack et al. (1980) Pharm. Ther. 8: 629-665. DPD deficiency is associated with inherited disorders of pyrimidine metabolism, clinically termed thymine-uraciluria. See, Bakkeren et al. (1984) Clin. chim. Acta. 140: 247-256. Clinical symptoms of DPD deficiency include a nonspecific cerebral dysfunction, and DPD deficiency is associated with psychomotor retardation, convulsions, and epileptic conditions. See, Berger et al. (1984) Clin. Chim. Acta 141: 227-234; Wadman et al. (1985) Adv. Exp. Med. Biol. 165A: 109-114; Wulcken et al. (1985) J. Inherit. Metab. Dis. 8 (Suppl. 2): 115-116; van Gennip et al. (1989) Adv. Exp. Med. Biol. 253A: 111-118; Brockstedt et al. (1990) J. Inherit. Metab. Dis. 12: 121-124; and Duran et al. (1991) J. Inherit. Metab. Dis. 14: 367-370. Biochemically, patients having DPD deficiency have an almost complete absence of DPD activity in fibroblasts (see, Bakker et al., supra) and in lymphocytes (see, Berger et al., supra and Piper et al. (1980) Biochim. Biophys. Acta 633: 400-409. These patients( typically have a large accumulation of uracil and thymine in their cerebrospinal fluid (see, Bakkeren et al., supra.) and urine (see, Berger et al., supra.; Bakkeren et al., supra.; Brockstedt et al., supra.; and Fleming et al. (1992) Cancer Res. 52: 2899-2902).
Familial studies suggest that DPD deficiency follows an autosomal racessive pattern of inheritance. See, Diasio et al., (1988) supra. Up to three percent of the general human population are estimated to be putative heterozygotes for DPD deficiency, as determined by enzymatic activity in lymphocytes. See, Milano and Eteinne (1994) Pharmacogenetics. This suggests that the frequency of homozygotes for DPD deficiency may be as high as one person per thousand.
DPD has been purified from liver tissue of rats (see, Shiotani and Weber (1981) J. Biol. Chem. 256: 219-224; Fujimoto et al. (1991); J. Nutr. Sci. Vitaminol. 37:89-98], pig [Podschun et al. (1989) Eur. J. Biochem. 185: 219-224), cattle (see, Porter et al. (1991) J. Biol. Chem. 266: 19988-19994), and humans (see, Lu et al. (1992) J. Biol. Chem. 267: 1702-1709). The pig enzyme contains flavins and iron-sulfur prosthetic groups and exists as a homodimer with a monomer Mr of about 107,000 (see, Podschun et al., supra.). Since the enzyme exhibits a nonclassical two-site ping-pong mechanism, it appears to have distinct binding sites for NADPH/NADP and uracil/5,6-dihydrouracil (see, Podschun et al. (1990) J. Biol. Chem. 265: 12966-12972). An acid-base catalytic mechanism has been proposed for DPD (see, Podschun et al. (1993) J. Biol. Chem. 268: 3407-3413).
The DPD cDNA is described in copending U.S. application Ser. No. 08/304,309. Recently, DPD mRNA from patients lacking dihydrompyrimidine activity was found to lack an exon which encodes a 165 base pair sequence found in the wild-type DPD cDNA. See, Meinsma et al. (1995) DNA and Cell Biology 14(1): 1-6.
Because an undetected DPD deficiency poses a significant danger to a cancer patient who is being treated with 5-FU, a great need exists for a simple and accurate test for DPD deficiency. Such a test will also facilitate diagnosis of disorders that are associated with DPD deficiency, such as uraciluria. The present invention provides such a test, thus fulfilling these and other needs.
Particular mutations in the dihydropyrimidine dehydrogenase gene are described herein which lead to loss of dihydropyrimidine dehydrogenase activity. The mutations result in a loss of the amino acids from 581-635 of the protein encoded by the gene due to a splicing defect. The splicing defect results in the loss of an exon encoding the missing amino acids. The assays and compositions of the invention detect the splicing defect in the genomic DNA which results in the loss of the exon.
A variety of assays for detecting splicing defect mutations in patients are provided. The assays determine whether RNA encoded by genomic DNA is competent to be spliced to produce mRNA with nucleic acids encoding the exon which corresponds to amino acids from 581-635 of the wild-type protein. For example, sequencing, PCR, LCR, and oligonucleotide array based assays are used to detect the mutations.
In one class of embodiments, the methods comprise the step of determining whether a nucleic acid encoding an mRNA for the dihydropyrimidine dehydrogenase gene has an exon corresponding to amino acids 581-635 of a corresponding wild-type dihydropyrimidine dehydrogenase mRNA. This determination is performed in sequencing, PCR, LCR, and oligonucleotide array based hybridization assays. For example, in one class of PCR based assays, the intronic genomic DNA encoding the dihydropyrimidine dehydrogenase in the region flanking the exon which encodes amino acids 581-635 is hybridized to a set of PCR primers for amplification and analysis of the intron-exon splice boundaries.
Example primers which are used for amplifying the splice regions include DELF1 and DELR1. Similar primers which hybridize to the same sites, or to sites proximal to the primer binding sites are also used.
One particular mutation which results in an abnormal dihydropyrimidine dehydrogenase gene phenotype is the conversion of a G to an A residue at the 3xe2x80x2 GT splice site of the exon which encodes amino acids 581-635 of the corresponding wild-type protein. The 3xe2x80x2 wild-type splice site is recognized by restriction endonucleases which recognize the Mae II cleavage site. The ability of the cleavage site to be cleaved by restriction endonucleases which recognize the Mae II site is used to distinguish wild-type from abnormal dihydropyrimidine dehydrogenase genomic DNAs. For example, a region including the mutation is amplified by PCR, and the PCR products cleaved by Mae II. This results in the cleavage of nucleic acids amplified by the wild-type gene, but mutant sequences are not amplified.
In addition to PCR detection methods, nucleic acid arrays are used to discriminate single base-pair mismatches, or to directly sequence DNA by hybridization to arrays. The sequence of the splice site is also determined by standard or PCR sequencing of the site, e.g., using primers which flank the site in a pol I or taq PCR based sequencing assay.
In addition to methods and compositions for abnormal dihydropyrimidine dehydrogenase gene detection, the invention provides kits for practicing the methods. Typically, the kit contains a first PCR primer which binds to DNA 3xe2x80x2 of a splice site in the genomic DNA for dihydropyrimidine dehydrogenase gene for an exon encoding amino acids 581-635, and a second PCR primer which binds to DNA 5xe2x80x2 of a splice site in the genomic DNA for dihydropyrimidine dehydrogenase gene for an exon encoding amino acids 581-635. The kit optionally contains other components such as instructions for the detection of an abnormal dihydropyrimidine dehydrogenase gene, restriction enzymes such as Mae II, PCR or other in vitro amplification reagents (buffers, enzymes, salts and the like), oligonucleotide array detection chips and the like.